Society for Maternal‐Fetal Medicine Statement: Evaluation and management of cell‐free DNA screening for fetal red cell antigen genotype in alloimmunized and non‐alloimmunized pregnancies

Abstract

A substantial portion of RhD‐negative pregnant patients carry an RhD‐negative fetus and are not at risk of RhD alloimmunization. Knowing the fetal RhD genotype allows such patients to avoid unnecessary monitoring or treatment. The use of cell‐free DNA (cfDNA) to detect the fetal RhD genotype from maternal blood has been proposed to identify pregnancies in alloimmunized patients at risk for hemolytic disease of the fetus and newborn and to decrease the antenatal use of Rho(D) immune globulin (RhIg) in patients not at risk. However, a number of factors limit the utility of cfDNA for RhD genotyping, including a lack of data that adequately reflect US populations or US laboratory testing approaches and a lack of data or research from independent sources. This document describes considerations and offers guidance on cfDNA screening for red blood cell antigen genotypes in three scenarios: RhD in a non‐alloimmunized patient, RhD in a non‐alloimmunized patient in the setting of an RhIg shortage, and any fetal red cell antigen genotype in an alloimmunized obstetrical patient.

1. INTRODUCTION

In the United States, the standard of care for identifying patients at risk of red cell sensitization is blood typing and antibody screening in the first trimester of pregnancy. To prevent maternal sensitization and subsequent hemolytic disease of the fetus and newborn (HDFN), all pregnant people who are RhD‐negative and RhD antibody screen‐negative should receive prophylactic Rho(D) immune globulin (RhIg) at 26–28 weeks of gestation if their reproductive partner is confirmed as RhD‐positive or of unknown status. RhIg should also be given within 72 h of delivery if the newborn is confirmed to be RhD‐positive. RhIg may be administered antenatally to pregnant people who are RhD‐negative and RhD antibody screen‐negative at the time of potentially sensitizing events, such as fetal diagnostic testing, suspected placental abruption, miscarriage, or abortion, depending on gestational age and clinical context. There is no equivalent to RhIg for other red cell antigens.

Approximately 40% of RhD‐negative pregnant patients carry an RhD‐negative fetus and are not at risk of RhD alloimmunization [1]. In current obstetrical practice, knowledge of the fetal RhD genotype enables patients not at risk for alloimmunization and HDFN to avoid unnecessary monitoring, treatments, and preventive strategies [2]. The RhD genotype refers to the genetic sequence that encodes the RhD antigen, a protein found on the surface of red blood cells. RhD phenotype refers to the observable physical expression of the genotype, which, in this case, is the presence or absence of the RhD antigen on the surface of red blood cells. A blood type report will document the RhD phenotype. The presence of at least one D allele results in the expression of the RhD antigen on the surface of red blood cells (i.e., the RhD‐positive phenotype). Testing for the RhD genotype is clinically available.

Data regarding the accuracy of cell‐free DNA (cfDNA) for fetal RhD and other red cell antigen genotyping are rapidly evolving. This document addresses the use of cfDNA screening for red blood cell antigen genotypes in three scenarios: RhD in a non‐alloimmunized patient, RhD in a non‐alloimmunized patient in the setting of an RhIg shortage, and any fetal red cell antigen genotype in an alloimmunized obstetrical patient.

1.1. Current evaluation of the alloimmunized pregnant person

If a pregnant patient is RhD‐negative and alloimmunized (RhD antibody screen‐positive), RhD zygosity testing using genotypic analysis of the reproductive partner (or sperm donor) is recommended, if available [3]. If the reproductive partner is heterozygous for RhD, amniocentesis is recommended to determine the fetal RhD genotype. If the fetus is confirmed to be RhD antigen‐positive, or if the fetal RhD status is unknown because amniocentesis was declined, routine surveillance for HDFN includes serial antibody titers, with Doppler assessments of the middle cerebral artery (MCA) peak systolic velocity once a critical titer is reached to identify risk for moderate to severe fetal anemia [4, 5]. If the reproductive partner is homozygous for RhD, no further fetal RhD genotype testing is indicated, as the fetus is an obligate heterozygote (RhD‐positive) and should undergo routine surveillance for HDFN. In situations in which the fetal RhD status is unknown and the reproductive partner is either heterozygous for RhD or unavailable for testing, the pregnancy is considered at risk of HDFN. Antibody titers and MCA Doppler surveillance are recommended in these scenarios, and, as a result, some undiagnosed RhD‐negative fetuses may be exposed to the risks of false‐positive MCA Doppler assessments, including unnecessary percutaneous umbilical blood sampling or early delivery. This same recommendation applies to other clinically significant red cell antigens [4].

1.2. cfDNA for fetal RhD genotype

The use of cfDNA to detect the fetal RhD genotype from maternal blood has been proposed to identify pregnancies in alloimmunized patients at risk for HDFN and to decrease the antenatal use of RhIg among non‐alloimmunized RhD‐negative patients with RhD‐negative fetuses. It is important to recognize that cfDNA assays are purported to identify antigen genotypes known to be associated with fetal anemia. However, our current understanding of all potential genotypes that may predispose to fetal anemia is incomplete [6]. This limitation is particularly pronounced in individuals from underrepresented ancestral backgrounds, for whom genetic variability may not be fully captured by existing literature and assays. Thus, there is a risk for false‐negative, false‐positive, or uninterpretable results in these populations, underscoring the need for cautious interpretation and robust clinical validation and utilization data [7].

Early guidance from the Society for Maternal‐Fetal Medicine on cfDNA for RhD genotyping [4] was based on data from a now‐closed commercial laboratory that used polymerase chain reaction (PCR)‐based technology no longer available in the United States. While several European countries consider PCR‐based cfDNA testing standard of care, the specific assays used differ from those currently available in the United States, limiting the direct applicability of international data to domestic practice [7, 8]. A 2017 meta‐analysis evaluated 30 studies involving cfDNA testing for fetal RhD status. Although the analysis showed a high pooled sensitivity and specificity, the evidence is limited by substantial heterogeneity and frequent indeterminate results. In nearly half of the studies (13/30), inconclusive results were reported, most often due to unspecified causes, suspected or confirmed RhD variants, insufficient marker counts, assay failure, or presumed low fetal fraction [9]. A recent meta‐analysis, which included most of these same studies, also aimed to evaluate the performance of cfDNA tests for fetal red blood cell antigen genotyping. It is notable that 79 of the 84 included studies in this analysis used PCR‐based noninvasive prenatal testing methods, whereas US laboratories that currently offer testing use next‐generation sequencing (NGS) technology [8]. Study sample sizes for non‐RhD red blood cell antigens were notably small, and variability in laboratory techniques limited cross‐study comparability. Further, negative results may require additional molecular confirmation, particularly in genetically diverse populations for whom serology testing may be less reliable. Understanding differences in laboratory methodologies and their impact on test performance and validation across diverse populations is essential for accurate assessment of clinical utility, validity, and equitable clinical application.

One industry‐sponsored peer‐reviewed publication presented proof‐of‐concept clinical data to support the validity and utility of a different methodology, NGS cfDNA with quantitative counting templates, to identify the fetal RhD genotype. This clinical validation included 23 RhD‐negative pregnant individuals and excluded two, one whose sample did not pass quality metrics due to low fetal fraction (i.e., a nonreportable or “no call” result) and one with conflicting clinical serology results [10]. A second prospective cohort study from this same commercial laboratory compared cfDNA fetal RhD genotyping to a gold standard of neonatal RhD genotyping among 155 alloimmunized pregnant patients and their neonates [11]. Of those, 41 pregnant patients were alloimmunized to RhD, and there was 100% concordance between cfDNA analysis of fetal RhD status and the neonatal genotype. In this study, all samples had fetal RhD determinations, and one sample was excluded from the concordance analysis due to inconclusive neonatal RhD genotyping. A third retrospective cohort study from this commercial laboratory evaluated the performance of a cfDNA fetal RhD molecular assay in non‐alloimmunized RhD‐negative patients and demonstrated 100% concordance between cfDNA RhD results and neonatal serologies in 401 dyads [12].

A second commercial laboratory published clinical validation data using samples from 655 RhD‐negative pregnant patients submitted for NGS‐based aneuploidy screening [13]. Using postnatal RhD testing as the gold standard, cfDNA correctly identified all 356 RhD‐positive fetuses and 295 of 297 RhD‐negative fetuses (two RhD‐negative fetuses were identified as RhD‐positive). Two of the 655 samples did not receive a result (i.e., nonreportable) because they did not pass quality metrics. Although both validation studies attempted to account for genomic diversity by including participants from Black, Asian, and Hispanic populations, overall representation remained limited. Across both cohorts, only 60 individuals who identified as Black and 21 as Asian were included. For population‐based national recommendations to be robust, it is imperative that study populations adequately reflect the genomic diversity inherent in red blood cell antigen genotypes.

Although the data are encouraging, limitations of these studies include moderate sample size, lack of non‐industry data or research involving independent investigators without monetary investment or other conflicts of interest, and underrepresentation of individuals from diverse ancestral backgrounds [14]. Data regarding cfDNA for red blood cell antigen screening in multifetal pregnancies are extremely limited [11].

1.3. RhD nondeletion genotype

In most patients, the RhD‐negative genotype results from a complete deletion of the RhD gene on chromosome 1. Advanced molecular technologies, including sequencing, have subsequently revealed the complexity of RhD genotypes. Hundreds of unique pathogenic variants and copy number variations within the RhD gene can also lead to an RhD‐negative phenotype [15]. These “nondeletion genotypes” (sometimes referred to as “weak RhD” variants) distinguish cases in which the RhD gene is altered but not fully deleted. PCR‐based methods may be inaccurate in predicting D phenotype, particularly in populations with a significant proportion of individuals of African ancestry. Nondeletion genotypes, such as the RhD pseudogene [16], are more commonly observed in these populations [17]. Current published data on cfDNA validation for nondeletion RhD genotypes are limited, and rates of false‐positive and false‐negative fetal RhD genotype predictions by cfDNA screening are unknown [13]. Further research is needed to evaluate cfDNA performance for RhD genotyping in diverse populations.

1.4. Laboratory development of cfDNA screening

In the United States, cfDNA screening falls into the US Food and Drug Administration (FDA) category of Laboratory Developed Tests (LDTs), which are both developed and performed within a single laboratory certified under the Clinical Laboratory Improvement Amendments (CLIA) program. The Centers for Medicare & Medicaid Services oversees LDTs under CLIA rules, which govern laboratory quality but not the clinical validity of the tests themselves. This approach allowed cfDNA companies to bring their tests to market without a lengthy and expensive FDA premarket review process and to market them directly to obstetrical providers. As cfDNA screening and LDTs became more widespread and complex, the FDA raised concerns, highlighting the risk to patients of false‐positive and false‐negative results [18]. A 2024 effort to classify LDTs as medical devices and thereby improve oversight was blocked [19]. As the data and uses of these tests are considered, it is important to recognize the inherent differences in the regulation and oversight of cfDNA tests compared with previous reproductive genetic screening modalities.

At the time of this publication, two commercial laboratories in the United States offer cfDNA screening for fetal RhD genotyping in conjunction with common aneuploidy cfDNA screening (i.e., neither commercial laboratory offers cfDNA fetal RhD genotype as a standalone test). Currently, only one laboratory offers testing for other red blood cell antigens. Unlike RhD, which is routinely assessed in every pregnancy through standard blood typing, patients with alloimmunization to other red blood cell antigens would come to attention only with a positive antibody screen. At present, there is no standardized or transparent mechanism to ensure that false‐negative or false‐positive results from cfDNA screening are consistently reported to laboratories and subsequently incorporated into future research and data analyses. As a result, clinically observed discrepancies are unlikely to be reflected in aggregated datasets, limiting the accuracy and completeness of evidence used to inform clinical practice [20]. In a recent Delphi‐based consensus document, content experts suggested that cfDNA for red cell genotype can be used as a diagnostic test. However, the authors noted that the accuracy of cfDNA screening results for red cell antigens from available studies may have been overestimated due to methodological and study design limitations. No studies were identified that directly compared the risks and benefits of using cfDNA for red cell genotyping with standard management. Economic analysis conducted in the United States found that using cfDNA to guide RhIg administration is not cost‐effective when compared with routine universal RhIg prophylaxis, although this evaluation did not account for RhIg supply constraints [21]. Overall, the available evidence is insufficient and too methodologically heterogeneous to support routine use of cfDNA for red blood cell antigen genotype as a diagnostic test. The evolving landscape of cfDNA screening underscores the importance of continued rigorous research and ongoing multidisciplinary dialogue among clinicians, laboratories, patients, and other stakeholders to inform equitable and evidence‐based practice.

1.5. RhIg shortages

Beginning in 2023, some practice settings experienced RhIg shortages [22], increasing interest in the use of cfDNA for fetal RhD genotyping in non‐alloimmunized pregnancies to avoid administration of RhIg in pregnancies with fetuses predicted to be RhD‐negative. A March 2024 American College of Obstetricians and Gynecologists Practice Advisory suggested that using cfDNA to determine fetal RhD status to prioritize and conserve RhIg is a “reasonable consideration in the practice setting that is experiencing RhIg shortages.” [23]

2. GUIDANCE FOR cfDNA SCREENING FOR FETAL RED BLOOD CELL ANTIGEN GENOTYPE

2.1. General clinical principles

• In clinical settings where RhIg is readily available, cfDNA screening for fetal RhD genotype is not currently recommended for the non‐alloimmunized patient. Obstetrical management should continue to follow established standard practice guidelines [2, 3, 4, 5]. Updated cost‐effectiveness analyses relevant to implementation of cfDNA for non‐alloimmunized pregnancies in the United States are warranted, along with robust data addressing the clinical implications and recommended management strategies for inconclusive or nonreportable cfDNA RhD results.

• cfDNA for fetal red blood cell antigen genotype is not recommended in a multifetal pregnancy given the lack of data.

• cfDNA for fetal red blood cell antigen genotype other than RhD is not recommended, given the limited data and that these patients are already alloimmunized. If, after counseling on the limitations of the test, a patient elects to proceed with red blood cell antigen genotyping using cfDNA, this can be considered in shared decision‐making.

• cfDNA screening for fetal red blood cell antigen genotype is not indicated when the reported reproductive partner or sperm donor is known to be homozygous for the red blood cell antigen of interest, as the fetal antigen phenotype is expected to be antigen‐positive (obligate heterozygote).

• Providers offering cfDNA screening for fetal RhD genotyping (see below for specific clinical scenarios) should first confirm that the pregnancy is eligible for cfDNA screening for fetal RhD genotyping (e.g., singleton pregnancy, non‐weak‐D antigen, no history of previous pregnancy with HDFN). Based on available data, cfDNA screening for fetal RhD genotype among patients with weak RhD variant/RhD nondeletion genotypes (if known previously) or among patients with alloimmunized pregnancies is not recommended.

• Pregnant patients who are potentially eligible for cfDNA screening for fetal RhD genotyping (see below for specific clinical scenarios) should have pretest counseling (see Box 1).

• Patients who undergo cfDNA screening for fetal RhD genotyping and receive aneuploidy screening results discordant from those previously reported should be referred for genetic counseling or for consultation with a maternal‐fetal medicine subspecialist or both.

• Patients who undergo cfDNA screening for fetal RhD genotyping and receive a “nonreportable result” or “result of uncertain significance” for fetal RhD status should be referred to a maternal‐fetal medicine subspecialist for counseling and consideration of diagnostic testing [4, 5].

• Manufacturers should develop mechanisms by which patients and providers may report unexpected or adverse outcomes related to cfDNA screening and make this information available in a transparenty fashion.

BOX 1. Pretest counseling for limitations of cfDNA screening for red cell genotype

Clinicians should document that patients have been counseled about the following points:

• Risk of false‐negative result when using cfDNA‐based assays for fetal red blood cell genotyping.

• Potential for significant fetal morbidity or mortality in cases in which a false‐negative result fails to identify an at‐risk, antigen‐positive fetus.

• Financial considerations, including the cost of testing and variability in insurance reimbursement.

• Possibility of obtaining a nonreportable result or a result of uncertain clinical significance.

• Commercial assays integrate fetal red blood cell genotyping with concurrent aneuploidy screening. Patients who have previously undergone aneuploidy testing may be exposed to the risk of receiving inconsistent or conflicting aneuploidy results.

2.2. Management of non‐alloimmunized RhD‐negative patients in the setting of RhIg shortage

• In practice settings facing RhIg shortages, cfDNA screening for fetal RhD genotype may be offered to non‐alloimmunized RhD‐negative patients, particularly when the RhD genotype of the reproductive partner or sperm donor is either heterozygous for the D antigen or unknown. Traditional management of non‐alloimmunized pregnancies should be offered first [2, 5]. Patients should undergo pretest counseling (see Box 1).

• In practice settings experiencing RhIg shortages, RhIg may be withheld in cases of an RhD‐negative patient with an RhD‐negative fetal genotype by cfDNA screening. If cfDNA screening predicts an RhD‐positive fetal genotype in a non‐alloimmunized patient who experiences a possibly sensitizing event, RhIg should be administered unless the shortage is so severe that RhIg doses are being conserved for third‐trimester or postpartum administration.

• Providers should engage in shared decision‐making to establish an evaluation and management strategy, understanding that evidence is currently lacking to support a clear best approach, and document informed consent for cfDNA for RhD testing if this strategy is selected.

2.3. Management of alloimmunized patients

• For an alloimmunized patient whose reproductive partner or sperm donor is heterozygous for red blood cell antigen or not available for testing, amniocentesis remains the standard of care to evaluate the fetal genotype. Amniocentesis has a lower risk of sensitization via fetal‐maternal hemorrhage than chorionic villus sampling [2, 5].

• If the patient declines diagnostic testing or empiric monitoring for HDFN, cfDNA screening can be offered to evaluate the fetal genotype, following pretest counseling (see Box 1) [3]. In the context of shared decision‐making, providers should outline the limitations, risks, and benefits of cfDNA screening compared with traditional screening methods for HDFN.

• An alloimmunized patient with an antigen‐positive fetus or inconclusive results upon cfDNA screening should have routine HDFN monitoring according to existing guidelines [5].

• Insufficient evidence exists to guide the management of alloimmunized patients with presumed red cell antigen‐negative fetuses by cfDNA screening. Shared decision‐making is therefore recommended in these cases to determine whether to conduct HDFN surveillance and, if so, at what frequency.

• Given the lack of published data evaluating the test performance of cfDNA among alloimmunized populations, especially with a history of HDFN, any alloimmunized patient with a critical titer or a previous pregnancy or newborn affected by HDFN should be managed according to established clinical guidance for at‐risk fetuses in pregnancy [5]. If, after counseling regarding the limitations of the testing, a patient declines diagnostic testing and elects to proceed with red blood cell antigen genotyping through cfDNA, this approach can be considered through shared decision‐making. The discussion should address the limitations, risks, and benefits of standard management (e.g., a false‐positive MCA Doppler result, costs) versus the consequences of undetected severe fetal anemia should the fetus in fact be antigen‐positive.

3. CONCLUSION

In practice settings experiencing RhIg shortages, cfDNA screening for fetal RhD genotype is a reasonable consideration in non‐alloimmunized RhD‐negative patients after comprehensive counseling. In RhD‐alloimmunized pregnancies, diagnostic testing remains the standard of care to evaluate the fetal genotype. cfDNA screening is not recommended in RhD‐alloimmunized pregnancies in patients with a pregnancy previously affected by HDFN, patients with other red blood cell alloantibodies, or in patients known to have a weak RhD variant/RhD nondeletion genotype. cfDNA screening for fetal red blood cell antigen genotyping should be available as a standalone test and not automatically combined with fetal aneuploidy screening due to limited insurance coverage and concern for discrepant results arising from multiple aneuploidy screening tests in the same pregnancy. Finally, given concerns regarding cfDNA accuracy among populations with RhD nondeletion genotypes, diverse populations should be prioritized in future research. We encourage further independent investigator‐initiated research regarding clinical application, validity, and cost analysis to inform the role of cfDNA screening for any red blood cell genotype. Manufacturers should develop mechanisms by which patients and providers may report unexpected or adverse outcomes related to cfDNA screening and make this information available in a transparent fashion. The Society for Maternal‐Fetal Medicine recognizes that this area in maternal‐fetal medicine is rapidly evolving, and data published after this guidance may influence clinical management.

4.

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